U.S. patent application number 10/446154 was filed with the patent office on 2004-01-15 for process for the preparation of l-amino acids using coryneform bacteria which contain an attenuated mez gene.
This patent application is currently assigned to Degussa AG. Invention is credited to Bathe, Brigitte, Farwick, Mike, Marx, Achim, Pfefferle, Walter.
Application Number | 20040009578 10/446154 |
Document ID | / |
Family ID | 29414265 |
Filed Date | 2004-01-15 |
United States Patent
Application |
20040009578 |
Kind Code |
A1 |
Bathe, Brigitte ; et
al. |
January 15, 2004 |
Process for the preparation of L-amino acids using coryneform
bacteria which contain an attenuated mez gene
Abstract
The present invention relates to a process for the preparation
of L-amino acids by the fermentation of coryneform bacteria in
which the gene which codes for the malate enzyme is attenuated.
Optionally, genes of the biosynthesis pathway of the desired
L-amino acid may be enhanced in the bacteria or metabolic pathways
which reduce the formation of the desired L-amino acid may be
diminished.
Inventors: |
Bathe, Brigitte;
(Salzkotten, DE) ; Farwick, Mike; (Essen, DE)
; Marx, Achim; (Banska Bystrica, SK) ; Pfefferle,
Walter; (Halle, DE) |
Correspondence
Address: |
Michael A. Sanzo
Fitch, Even, Tabin & Flannery
Suite 401L
1801 K Street, N.W.
Washington
DC
20006-1201
US
|
Assignee: |
Degussa AG
|
Family ID: |
29414265 |
Appl. No.: |
10/446154 |
Filed: |
May 28, 2003 |
Current U.S.
Class: |
435/252.3 |
Current CPC
Class: |
C12P 13/08 20130101;
C12P 13/12 20130101 |
Class at
Publication: |
435/252.3 |
International
Class: |
C12N 001/20 |
Foreign Application Data
Date |
Code |
Application Number |
May 31, 2002 |
DE |
102 24 088.4 |
Claims
What is claimed is:
1. A process for the preparation of a desired L-amino acid
comprising: a) fermenting modified coryneform bacteria to produce a
fermentation broth, wherein said modified coryneform bacteria
produce said desired L-amino acid, and wherein the nucleotide
sequence which codes for the malate enzyme (mez) in said modified
coryneform bacteria is attenuated relative to unmodified or
wild-type coryneform bacteria; b) concentrating said desired
L-amino acid in the fermentation broth or in the cells of said
coryneform bacteria of step a); and c) obtaining an isolated
composition by purifying the desired L-amino acid concentrated in
step b).
2. The process of claim 1, wherein said desired L-amino acid is
L-lysine.
3. The process of claim 1, wherein said desired L-amino acid is
L-methionine.
4. The process of claim 1, wherein the isolated composition
produced in step c) of said process comprises, in addition to said
desired amino acid, biomass from the fermentation of said modified
coryneform bacteria or constituents from said fermentation
broth.
5. The process of claim 1, wherein the activity of at least one
gene product in the biosynthetic pathway of said desired L-amino
acid is enhanced relative to the activity of the corresponding gene
product in the wild type coryneform bacteria.
6. The process of claim 5, wherein the expression of the gene
encoding said gene product is increased in said modified coryneform
bacteria relative to expression of the corresponding gene in the
wild type coryneform bacteria.
7. The process of claim 1, wherein at least one gene product in a
metabolic pathway that reduces the formation of said desired amino
acid is decreased in activity in said modified coryneform bacteria
relative to the corresponding gene product in the wild type
coryneform bacteria.
8. The process of claim 7, wherein the expression of the gene
encoding said gene product is reduced in said modified coryneform
bacteria relative to the corresponding gene in the wild type
coryneform bacteria.
9. The process of claim 1, wherein the expression of the
polynucleotide which codes for the malate enzyme is reduced.
10. The process of claim 1, wherein the catalytic properties of the
polypeptide for which the polynucleotide mez codes are reduced.
11. The process of claim 1, wherein said desired L-amino acid is
either L-lysine or L-methionine and, relative to wild type
coryneform bacteria, said modified coryneform bacteria exhibit
increased enzymatic activity or concentration of a protein selected
from the group consisting of: feed-back resistant aspartate kinase,
coded for by the lysC gene; dihydrodipicolinate synthase coded for
by the dapA gene; glyceraldehyde 3-phosphate dehydrogenase, coded
for by the gap gene; pyruvate carboxylase, coded for by the pyc
gene; malate:quinone oxidoreductase, coded for by the mqo gene;
glucose 6-phosphate dehydrogenase, coded for by the zwf gene; the
lysine export protein, coded for by the lysE; the Zwa1 protein,
coded for by the zwa1 gene; triose phosphate isomerase, coded for
by the tpi gene; and 3-phosphoglycerate kinase, coded for by the
pgk gene.
12. The process of claim 1 wherein said desired L-amino acid is
either L-lysine or L-methionine and, relative to wild type
coryneform bacteria, said modified coryneform bacteria overexpress
at least one gene selected from the group consisting of: the lysC
gene which codes for a feed-back resistant aspartate kinase; the
dapA gene which codes for dihydrodipicolinate synthase; the gap
gene which codes for glyceraldehyde 3-phosphate dehydrogenase; the
pyc gene which codes for pyruvate carboxylase; the mqo gene which
codes for malate:quinone oxidoreductase; the zwf gene which codes
for glucose 6-phosphate dehydrogenase; the lysE gene which codes
for the lysine export protein; the zwa1 gene which codes for the
Zwa1 protein; the tpi gene which codes for triose phosphate
isomerase; and the pgk gene which codes for 3-phosphoglycerate
kinase.
13. The process of claim 1, wherein said desired L-amino acid is
either L-lysine or L-methionine and, relative to wild type
coryneform bacteria, said modified coryneform bacteria exhibits
decreased enzymatic activity or concentration of a protein selected
from the group consisting of: phosphoenolpyruvate carboxykinase,
coded for by the pck gene; glucose 6-phosphate isomerase, coded for
by the pgi gene; pyruvate oxidase, coded for by the poxB gene; the
Zwa2 protein coded for by the zwa2 gene; and catabolite control
protein A, coded for by the ccpA1 gene.
14. The process of claim 1, wherein said desired L-amino acid is
either L-lysine or L-methionine and, relative to wild type
coryneform bacteria, at least one gene in said modified coryneform
bacteria is attenuated, said attenuated gene being selected from
the group consisting of: the pgi gene which codes for glucose
6-phosphate isomerase; the poxB gene which codes for pyruvate
oxidase; the zwa2 gene which codes for the Zwa2 protein; and the
ccpA1 gene which codes for a catabolite control protein A.
15. The process according of any one of claims 1-14, wherein said
modified coryneform bacteria and said wild type coryneform bacteria
are of the species Corynebacterium glutamicum.
16. The process of claim 15, wherein said coryneform bacteria of
the species Corynebacterium glutamicum comprise the plasmid
pK18mobsacBdeltamez.
17. A modified coryneform bacterium comprising a polynucleotide
coding for the malate enzyme (mez) that is attenuated relative to
unmodified or wild-type coryneform bacteria.
18. The modified coryneform bacterium of claim 17, wherein the
expression of the polynucleotide which codes for the malate enzyme
is reduced.
19. The modified coryneform bacterium of claim 17, wherein the
catalytic properties of the polypeptide for which the
polynucleotide mez codes are reduced.
20. The modified coryneform bacterium of claim 17, wherein,
relative to wild type coryneform bacteria, said modified coryneform
bacterium exhibits increased enzymatic activity or concentration of
a protein selected from the group consisting of: feed-back
resistant aspartate kinase, coded for by the lysC gene;
dihydrodipicolinate synthase coded, for by the dapA gene;
glyceraldehyde 3-phosphate dehydrogenase, coded for by the gap
gene; pyruvate carboxylase, coded for by the pyc gene;
malate:quinone oxidoreductase, coded for by the mqo gene; glucose
6-phosphate dehydrogenase, coded for by the zwf gene; the lysine
export protein, coded for by the lyse; the Zwa1 protein, coded for
by the zwa1 gene; triose phosphate isomerase, coded for by the tpi
gene; and 3-phosphoglycerate kinase, coded for by the pgk gene.
21. The modified coryneform bacterium of claim 17, wherein relative
to wild type coryneform bacteria, said modified coryneform
bacterium overexpresses at least one gene selected from the group
consisting of: the lysC gene which codes for a feed-back resistant
aspartate kinase; the dapA gene which codes for dihydrodipicolinate
synthase; the gap gene which codes for glyceraldehyde 3-phosphate
dehydrogenase; the pyc gene which codes for pyruvate carboxylase;
the mqo gene which codes for malate:quinone oxidoreductase; the zwf
gene which codes for glucose 6-phosphate dehydrogenase; the lysE
gene which codes for the lysine export protein; the zwa1 gene which
codes for the Zwa1 protein; the tpi gene which codes for triose
phosphate isomerase; and the pgk gene which codes for
3-phosphoglycerate kinase.
22. The modified coryneform bacterium of claim 17, wherein relative
to wild type coryneform bacteria, said modified coryneform
bacterium exhibits decreased enzymatic activity or concentration of
a protein selected from the group consisting of:
phosphoenolpyruvate carboxykinase, coded for by the pck gene;
glucose 6-phosphate isomerase, coded for by the pgi gene; pyruvate
oxidase, coded for by the poxB gene; the Zwa2 protein coded for by
the zwa2 gene; and catabolite control protein A, coded for by the
ccpA1 gene.
23. The modified coryneform bacterium of claim 17, wherein,
relative to wild type coryneform bacteria, at least one gene in
said modified coryneform bacterium is attenuated, said attenuated
gene being selected from the group consisting of: the pgi gene
which codes for glucose 6-phosphate isomerase; the poxB gene which
codes for pyruvate oxidase; the zwa2 gene which codes for the Zwa2
protein; and the ccpA1 gene which codes for a catabolite control
protein A.
24. The modified coryneform bacterium of any one of claims 17-23,
wherein said modified coryneform bacterium and said wild type
coryneform bacteria are of the species Corynebacterium
glutamicum.
25. The modified coryneform bacterium of claim 17, wherein said
modified coryneform bacterium of the species Corynebacterium
glutamicum comprise the plasmid pK18mobsacBdeltamez.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims priority to German
application 102 24 088.4, filed on May 31, 2002, the contents of
which is hereby incorporated by reference.
FIELD OF THE INVENTION
[0002] The invention relates to a process for the preparation of
L-amino acids using coryneform bacteria in which the mez gene,
which codes for the malate enzyme (malic enzyme, EC:1.1.1.40), is
attenuated.
BACKGROUND OF THE INVENTION
[0003] L-amino acids, especially L-lysine and L-methionine, are
used in animal nutrition, in human medicine and in the
pharmaceuticals industry. One effective manner of producing amino
acids for these purposes is by the fermentation of strains of
coryneform bacteria and, in particular, Corynebacterium glutamicum.
Because of its great importance, improvements are constantly being
made in this process. Such improvements may relate to fermentation
procedures (e.g., the stirring of preparations or supply of oxygen)
or to the composition of the nutrient media (e.g., the sugar
concentration present during fermentation). Alternatively,
improvements may relate to the methods by which product is purified
or to the intrinsic synthetic properties of the microorganism
itself.
[0004] Methods of mutagenesis and selection have been used to
increase the amount of amino acid produced by microorganisms.
Strains which are resistant to antimetabolites, e.g., the lysine
analogue S-(2-aminoethyl)-L-cysteine or that are auxotrophic for
metabolites of regulatory importance and produce L-amino acids may
be obtained in this manner. In addition, recombinant DNA techniques
have been used to improve the production characteristics of
Corynebacterium glutamicum strains.
OBJECT OF THE INVENTION
[0005] The object of the present invention is to provide improved
procedures for the fermentative preparation of L-amino acids,
particularly L-lysine and L-methionine, by coryneform bacteria.
SUMMARY OF THE INVENTION
[0006] The present invention is based upon the development of an
improved process for the preparation of L-amino acids by fermenting
coryneform bacteria. In this process, bacteria are used that have
been modified so that their nucleotide sequence coding for the
malate enzyme (malic enzyme; mez gene) is attenuated and, in
particular, eliminated or expressed at a low level. The malate
enzyme catalyses oxidative decarboxylation of malate to pyruvate,
with a molecule of carbon dioxide being split off. The invention
also encompasses the modified bacteria used in the processes.
[0007] In its first aspect, the invention is directed to a process
for producing a desired amino acid by fermenting coryneform
bacteria and thereby creating a fermentation broth. The bacteria
used have been modified so that their nucleotide sequence coding
for the malate enzyme (mez) is attenuated relative to the
unmodified or wild-type coryneform bacteria. In the next step, the
desired L-amino is concentrated in the fermentation broth or in the
fermented bacterial cells and is then purified to make an isolated
composition which may optionally include, in addition to the
desired amino acid, biomass or constituents from the fermentation
broth.
[0008] As used herein, the term "L-amino acids" or "amino acids"
means one or more amino acids, including their salts, chosen from
the group consisting of L-asparagine, L-threonine, L-serine,
L-glutamate, L-glycine, L-alanine, L-cysteine, L-valine,
L-methionine, L-isoleucine, L-leucine, L-tyrosine, L-phenylalanine,
L-histidine, L-lysine, L-tryptophan and L-arginine. A "desired
amino acid" is one which the user of the disclosed methods wishes
to produce, with L-lysine and L-methionine being particularly
preferred. When the terms "L-lysine" or "lysine" are mentioned
herein, they will be understood to refer not only to the bases, but
also to salts of the amino acids, such as, e.g., lysine
monohydrochloride or lysine sulfate. Similarly, when "L-methionine"
or "methionine" are mentioned, this includes salts such as, e.g.,
methionine hydrochloride and methionine sulfate.
[0009] In alternative embodiments, the bacteria used in
fermentations may contain modifications in addition to those
affecting the gene for the malate enzyme as described above. For
example, the bacteria may be modified so that at least one gene
product in the biosynthetic pathway of the desired L-amino acid is
enhanced, i.e., increased, relative to the activity of the
corresponding gene product in the wild type or unmodified,
coryneform bacteria, e.g., by increasing expression of the
corresponding gene. Alternatively, a gene product in a metabolic
pathway that reduces the formation of the desired amino acid may be
reduced in activity in the modified coryneform bacteria. This may
be accomplished either by reducing the protein's biological
activity or by reducing gene expression of the protein.
[0010] In one set of preferred embodiments, the desired L-amino
acid is either L-lysine or L-methionine and, relative to wild type
or unmodified coryneform bacteria, the modified coryneform bacteria
have an increased enzymatic activity or concentration of one or
more of the following proteins: feed-back resistant aspartate
kinase, coded for by the lysC gene; dihydrodipicolinate synthase,
coded for by the dapA gene; glyceraldehyde 3-phosphate
dehydrogenase, coded for by the gap gene; pyruvate carboxylase,
coded for by the pyc gene; malate:quinone oxidoreductase, coded for
by the mqo gene; glucose 6-phosphate dehydrogenase, coded for by
the zwf gene; the lysine export protein, coded for by the lyse; the
Zwa1 protein, coded for by the zwa1 gene; triose phosphate
isomerase, coded for by the tpi gene; and 3-phosphoglycerate
kinase, coded for by the pgk gene. In each case, protein activity
can be increased by overexpressing the gene encoding the
protein.
[0011] In an alternative set of preferred embodiments, the desired
L-amino acid is either L-lysine or L-methionine and, relative to
wild type coryneform bacteria, the modified coryneform bacteria
exhibit decreased enzymatic activity or concentration of one or
more of the following proteins: phosphoenolpyruvate carboxykinase,
coded for by the pck gene; glucose 6-phosphate isomerase, coded for
by the pgi gene; pyruvate oxidase, coded for by the poxB gene; the
Zwa2 protein coded for by the zwa2 gene; and catabolite control
protein A, coded for by the ccpA1 gene. One way to reduce activity,
is by reducing the expression of genes encoding the proteins.
[0012] In all cases, the most preferred modified coryneform
bacteria and wild type coryneform bacteria are of the species
Corynebacterium glutamicum. Corynebacterium glutamicum modified
using the plasmid pK18mobsacBdeltamez are of particular use in the
invention.
[0013] In a different but related aspect, the invention is directed
to a modified coryneform bacterium in which the polynucleotide
coding for the malate enzyme (mez) is attenuated relative to
unmodified or wild-type coryneform bacteria, preferably by reducing
mez gene expression. Relative to its wild type counterparts, the
modified bacterium may also exhibit increased enzymatic activity or
concentration of a protein from the group consisting of: feed-back
resistant aspartate kinase, coded for by the lysC gene;
dihydrodipicolinate synthase, coded for by the dapA gene;
glyceraldehyde 3-phosphate dehydrogenase, coded for by the gap
gene; pyruvate carboxylase, coded for by the pyc gene;
malate:quinone oxidoreductase, coded for by the mqo gene; glucose
6-phosphate dehydrogenase, coded for by the zwf gene; the lysine
export protein, coded for by the lysE; the Zwa1 protein, coded for
by the zwa1 gene; triose phosphate isomerase, coded for by the tpi
gene; and 3-phosphoglycerate kinase, coded for by the pgk gene. In
general, this increased activity or concentration can be achieved
by overexpressing the relevant gene.
[0014] As an alternative to, or in conjunction with, increasing the
activity of the proteins described above, the modified bacterium
may exhibit decreased enzymatic activity or concentration of a
protein selected from: phosphoenolpyruvate carboxykinase, coded for
by the pck gene; glucose 6-phosphate isomerase, coded for by the
pgi gene; pyruvate oxidase, coded for by the poxB gene; the Zwa2
protein coded for by the zwa2 gene; and catabolite control protein
A, coded for by the ccpA1 gene. These bacteria may be characterized
by decreased expression of the genes for one or more of these
proteins.
[0015] The most preferred bacterium is of the species
Corynebacterium glutamicum, with bacteria made using the plasmid
pK18mobsacBdeltamez being of particular use.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1: Map of the plasmid pK18mobsacBdeltamez: The base
pair numbers shown in the FIGURE are approximate values obtained in
the context of reproducibility of measurements. The abbreviations
and designations used have the following meaning:
1 oriV: ColE1-similar origin from pMB1; sacB: the sacB gene which
codes for the protein levan sucrase; RP4mob: RP4 mobilization site;
Km: resistance gene for kanamycin; LacZ': 5'-terminus of the
lacZ.alpha. gene fragment; LacZ: 3'-terminus of the lacZ.alpha.
gene fragment; deltamez: deleted allele of the mez gene from C.
glutamicum; BamHI: cleavage site of the restriction enzyme BamHI;
EcoRI: cleavage site of the restriction enzyme EcoRI.
DETAILED DESCRIPTION OF THE INVENTION
[0017] The term "attenuation" or "attenuate" as used herein
describes a reduction or elimination of the intracellular activity
or concentration of one or more enzymes or proteins in a
microorganism. This may be accomplished, for example, by putting
the gene encoding the protein under the control of a weak promoter,
by replacing the gene with a sequence that codes for a
corresponding enzyme with a low activity, by inactivating the gene,
by directly inactivating the protein, or, optionally, by combining
these measures. Using such measures, the activity or concentration
of the corresponding protein may be reduced to 0 to 75%, 0 to 50%,
0 to 25%, 0 to 10% or 0 to 5% of the activity or concentration of
the wild-type protein or of the activity or concentration of the
protein in the starting, i.e., unmodified, microorganism.
[0018] The microorganisms provided by the present invention can
prepare amino acids from glucose, sucrose, lactose, fructose,
maltose, molasses, starch, cellulose or from glycerol and ethanol.
They should preferably be coryneform bacteria of the genus
Corynebacterium. The most preferred species is Corynebacterium
glutamicum, which is known among experts for its ability to produce
L-amino acids. Suitable bacteria include the wild-type strains:
[0019] Corynebacterium glutamicum ATCC13032;
[0020] Corynebacterium acetoglutamicum ATCC15806;
[0021] Corynebacterium acetoacidophilum ATCC13870;
[0022] Corynebacterium melassecola ATCC17965;
[0023] Corynebacterium thermoaminogenes FERM BP-1539;
[0024] Brevibacterium flavum ATCC14067;
[0025] Brevibacterium lactofermentum ATCC13869; and
[0026] Brevibacterium divaricatum ATCC 14020.
[0027] L-lysine-producing mutants or strains that may be used
include:
[0028] Corynebacterium glutamicum FERM-P 1709;
[0029] Brevibacterium flavum FERM-P 1708;
[0030] Brevibacterium lactofermentum FERM-P 1712;
[0031] Corynebacterium glutamicum FERM-P 6463;
[0032] Corynebacterium glutamicum FERM-P 6464; and
[0033] Corynebacterium glutamicum DSM 5715.
[0034] An example of an L-methionine-producing strain that may be
used is Corynebacterium glutamicum ATCC21608.
[0035] The nucleotide sequence of the gene which codes for the
malate enzyme of Corynebacterium glutamicum can be found in the
patent application FR-A-2796080. This sequence has been deposited
in the databank of the National Center for Biotechnology
Information (NCBI) of the National Library of Medicine (Bethesda,
Md., USA) under Accession Number AF234535. The nucleotide sequence
of the gene has also been reported in patent application EP1108790
as sequence nos. 3328 and 7069, and in the patent application
WO0100844, see sequence no. 577 under identification code RXN10148.
These sequences are also deposited in the databank of the National
Center for Biotechnology Information (NCBI) of the National Library
of Medicine (Bethesda, Md., USA) under Accession Number AX123412,
under Accession Number AX127153 and under Accession Number
AX065451.
[0036] The patent application FR 2796080 suggests that by enhancing
the malate enzyme in Corynebacterium glutamicum, an increased
production of amino acids, in particular lysine, is achieved.
However, it has been found that, in direct contrast to the
teachings of this reference, coryneform bacteria produce L-amino
acids in an improved manner after the mez gene is attenuated. The
sequence of the mez gene from Corynebacterium glutamicum ATCC13032,
which codes for the malate enzyme, is shown in SEQ ID NO:1 and can
be used according to the invention. The amino acid sequence of the
protein is shown in SEQ ID NO:2. The nucleotide sequence of the mez
gene as set forth in SEQ ID NO:1 differs from the sequence
described in FR 2796080 at positions 138, 186, 351, 477, 483, 603,
606, 609, 612, 624, 627, 654, 657, 666, 719, 831, 837, 846, 849,
850, 1014, 1029, 1052 and 1111. The nucleobase differences at
position 719 and at position 850 of the nucleotide sequence lead to
changes in the amino acid sequence of the protein. Specifically, at
position 240 of the amino acid sequence, aspartate is present
instead of alanine and, at position 284 of the amino acid sequence,
glutamine is present instead of glutamate. Alleles of the malate
enzyme which result from the degeneracy of the genetic code or due
to "sense mutations" of neutral function can also be used in
connection with the present invention.
[0037] To achieve an attenuation, either the expression of the mez
gene and/or the catalytic properties of the gene product can be
reduced or eliminated. Gene expression can be reduced by suitable
culturing methods or by genetic modification (mutation) of the
signal structures affecting gene expression. These signal
structures include, for example, repressor sequences, activator
sequences, operators, promoters, attenuators, ribosome binding
sites, the start codon and terminators. One skilled in the art can
find a description of relevant methodology, e.g., in the patent
application WO 96/15246, in Boyd and Murphy (J. Bacteriol. 170:5949
(1988)), in Voskuil and Chambliss (Nucl. Acids Res. 26:3548
(1998)), in Jensen and Hammer (Biotech. Bioeng. 58:191 (1998)), in
Ptek et al. (Microbiol. 142:1297 (1999)) and in textbooks of
genetics and molecular biology, such as, e.g., the textbook by
Knippers ("Molekulare Genetik," 6th edition, Georg Thieme Verlag,
Stuttgart, Germany, 1995) or that by Winnacker ("Gene und Klone,"
VCH Verlagsgesellschaft, Weinheim, Germany, 1990).
[0038] Another method for reducing the expression of specific genes
is by the antisense technique, in which short oligodeoxynucleotides
or vectors for the synthesis of antisense RNA are brought into the
target cells. There, the antisense RNA can bind to complementary
sections of specific mRNAs and either reduce their stability or
block the rate at which they are translated. An example in this
context may be found in Srivastava, et al., (Appl. Environ.
Microbiol. 66(10):4366-4371 (2000)).
[0039] Mutations which lead to a change or reduction in the
catalytic properties of enzyme proteins have been described in the
prior art. Examples include disclosures by Qiu, et al., (J. Biol.
Chem. 272:8611-8617 (1997)), Sugimoto et al. (Biosci. Biotech.
Biochem. 61:1760-1762 (1997)) and Mockel ("Die Threonindehydratase
aus Corynebacterium glutamicum: Aufhebung der allosterischen
Regulation und Struktur des Enzyms", Reports from the Julich
Research Center, Jul-2906, ISSN09442952, Julich, Germany, 1994).
Summaries may also be found in textbooks of genetics and molecular
biology, such as, e.g., that by Hagemann ("Allgemeine Genetik",
Gustav Fischer Verlag, Stuttgart, 1986). Possible mutations are
transitions, transversions, insertions and deletions of at least
one base pair. Depending on the effect of the amino acid exchange
on the enzyme activity, "missense mutations" or "nonsense
mutations" are referred to. As a consequence of nonsense mutations,
sense codons are converted into stop codons and translation
terminates prematurely. Insertions or deletions of at least one
base pair in a gene can lead to "frame shift mutations," as a
consequence of which incorrect amino acids are incorporated or
translation is interrupted prematurely. Deletions of one or more
codons typically lead to a complete loss of enzyme activity.
Instructions on the generation of such mutations are well known in
the prior art and can be found in textbooks of genetics and
molecular biology, such as, e.g., the textbook by Knippers
("Molekulare Genetik", 6th edition, Georg Thieme Verlag, Stuttgart,
Germany, 1995), that by Winnacker ("Gene und Klone," VCH
Verlagsgesellschaft, Weinheim, Germany, 1990) or that by Hagemann
("Allgemeine Genetik", Gustav Fischer Verlag, Stuttgart, 1986).
[0040] Common methods of mutating genes of C. glutamicum include
the methods of "gene disruption" and "gene replacement" described
by Schwarzer, et al. (Bio/Technology 9:84-87 (1991)). In the method
of gene disruption, a central part of the coding region of the gene
of interest is cloned in a plasmid vector which can replicate in a
host (typically E. coli), but not in C. glutamicum. Possible
vectors are, for example: pSUP301 (Simon et al., Bio/Technology
1:784-791 (1983)); pK18mob or pK19mob (Schfer et al., Gene
145:69-73 (1994)); pK18mobsacB or pK19mobsacB (Jger, et al., J.
Bacteriol. 174:5462-65 (1992)); pGEM-T (Promega Corporation,
Madison, Wis., USA); pCR2.1-TOPO (Shuman, J. Biol. Chem.
269:32678-84 (1994), U.S. Pat. No. 5,487,993); pCR.RTM.Blunt
(Invitrogen, Groningen, Holland, Bernard et al., J. Mol. Biol.
234:534-541 (1993)); or pEM1 (Schrumpf, et al, J. Bacteriol.
173:4510-4516 (1991)). The plasmid vector which contains the
central part of the coding region of the gene is then transferred
into the desired strain of C. glutamicum by conjugation or
transformation. The method of conjugation is described, for
example, by Schfer et al. (Appl. Environ. Microbiol. 60:756-759
(1994)). Methods for transformation are described, for example, by
Thierbach et al. (Appl. Microbiol. Biotechnol. 29:356-362 (1988)),
Dunican, et al. (Bio/Technology 7:1067-1070 (1989)) and Tauch, et
al. (FEMS Microbiol. Lett. 123:343-347 (1994)). After homologous
recombination by means of a "cross-over" event, the coding region
of the gene in question is interrupted by the vector sequence and
two incomplete alleles are obtained, one lacking the 3' end and one
lacking the 5' end. This method has been used, for example, by
Fitzpatrick et al. (Appl. Microbiol. Biotechnol. 42:575-580 (1994))
to eliminate the recA gene of C. glutamicum.
[0041] In the method of "gene replacement," a mutation, such as,
e.g., a deletion, insertion or base exchange, is established in
vitro in the gene of interest. The allele prepared is cloned in a
vector which is not replicative for C. glutamicum and this is then
transferred into the desired C. glutamicum host by transformation
or conjugation. After homologous recombination by means of a first
"cross-over" event which effects integration and a suitable second
"cross-over" event which effects excision in the target gene or in
the target sequence, the incorporation of the mutation or of the
allele is achieved. This method was used, for example, by
Peters-Wendisch et al. (Microbiol. 144:915-927 (1998)) to eliminate
the pyc gene of C. glutamicum by a deletion. A deletion, insertion
or a base exchange can be incorporated into the mez gene using
these procedures.
[0042] In addition to the attenuation of the mez gene, it may be
advantageous for the production of L-amino acids to enhance, in
particular to overexpress, one or more enzymes of the biosynthesis
pathway of glycolysis, of anaplerosis, of the citric acid cycle, of
the pentose phosphate cycle, of amino acid export and, optionally,
regulatory proteins. The term "enhancement" or "enhance" in this
context describes an increase in the intracellular activity or
concentration of one or more enzymes or proteins in a microorganism
which are coded for by the corresponding DNA. Enhancement may be
accomplished, for example, by increasing the number of copies of
the gene or genes, by using a potent promoter, by using a gene or
allele which codes for a corresponding enzyme or protein with a
high activity, or, optionally, by combining these measures. Using
such methods the activity or concentration of the corresponding
protein may be increased by at least 10%, 25%, 50%, 75%, 100%,
150%, 200%, 300%, 400% or 500%, up to a maximum of 1000% or 2000%,
relative to that of the wild-type protein or to the activity or
concentration of the protein in the starting, microorganism.
[0043] The use of endogenous genes is, in general, preferred. The
terms "endogenous genes" or "endogenous nucleotide sequences" as
used herein are understood to refer to the genes or nucleotide
sequences present in the population of a species. Thus, for the
preparation of L-amino acids, in addition to attenuation of the mez
gene, one or more of the following genes can be enhanced, in
particular over-expressed:
[0044] the lysC gene which codes for a feed-back resistant
aspartate kinase (Accession No. P26512, EP-B-0387527; EP-A-0699759;
WO 00/63388);
[0045] the dapA gene which codes for dihydrodipicolinate synthase
(EP-B 0 197 335);
[0046] the gap gene which codes for glyceraldehyde 3-phosphate
dehydrogenase (Eikmanns, J. Bacteriol. 174:6076-6086 (1992));
[0047] the pyc gene which codes for pyruvate carboxylase
(EP-A-1083225);
[0048] the mqo gene which codes for malate:quinone oxidoreductase
(Molenaar et al., Eur. J. Biochem. 254:395-403 (1998);
EP-A-1038969);
[0049] the zwf gene which codes for glucose 6-phosphate
dehydrogenase (JP-A-09224661, WO 01/70995);
[0050] the lysE gene which codes for the lysine export protein
(DE-A-195 48 222);
[0051] the zwa1 gene which codes for the Zwa1 protein
(EP-A-1111062);
[0052] the tpi gene which codes for triose phosphate isomerase
(Eikmanns, J. Bacteriol. 174:6076-6086 (1992)); and
[0053] the pgk gene which codes for 3-phosphoglycerate kinase
(Eikmanns, J. Bacteriol. 174:6076-6086 (1992)).
[0054] It may furthermore be advantageous for the production of
L-amino acids, in addition to the attenuation of the mez gene, to
attenuate one or more of the following genes:
[0055] the pck gene which codes for phosphoenol pyruvate
carboxykinase (EP-A-1094111);
[0056] the pgi gene which codes for glucose 6-phosphate isomerase
(EP-A-1087015, WO 01/07626);
[0057] the poxB gene which codes for pyruvate oxidase
(EP-A-1096013);
[0058] the zwa2 gene which codes for the Zwa2 protein
(EP-A-1106693); and
[0059] the ccpA1 gene which codes for a catabolite control protein
A (WO 02/18419).
[0060] Finally, it may be advantageous for the production of amino
acids to eliminate undesirable side reactions (see Nakayama,
"Breeding of Amino Acid Producing Microorganisms," in:
Overproduction of Microbial Products, Krumphanzl, Sikyta, Vanek
eds., Academic Press, London, UK, 1982).
[0061] The invention also encompasses the microorganisms prepared
according to the methods described herein. These can be cultured
continuously or discontinuously in a batch process (batch culture),
in a fed batch (feed process) or in a repeated fed batch process
(repetitive feed process) for the purpose of producing L-amino
acids. A summary of culture methods is provided in the textbook by
Chmiel (Bioprozesstechnik 1. Einfuhrung in die
Bioverfahrenstechnik, Gustav Fischer Verlag, Stuttgart, 1991)) and
in the textbook by Storhas (Bioreaktoren und periphere
Einrichtungen, Vieweg Verlag, Braunschweig/Wiesbaden, 1994).
[0062] The culture medium to be used must meet the requirements of
the particular strains being fermented. Descriptions of culture
media for various microorganisms are contained in the handbook
"Manual of Methods for General Bacteriology" of the American
Society for Bacteriology (Washington D.C., USA, 1981). Sugars and
carbohydrates (e.g., glucose, sucrose, lactose, fructose, maltose,
molasses, starch and cellulose), oils and fats (e.g., soya oil,
sunflower oil, groundnut oil and coconut fat), fatty acids (e.g.,
palmitic acid, stearic acid and linoleic acid), alcohols (e.g.,
glycerol and ethanol), and organic acids (e.g., acetic acid) can be
used as the source of carbon. These substances can be used
individually or as a mixture.
[0063] Organic nitrogen-containing compounds, such as peptones,
yeast extract, meat extract, malt extract, corn steep liquor, soya
bean flour and urea, or inorganic compounds, such as ammonium
sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate
and ammonium nitrate, can be used as the source of nitrogen. The
sources of nitrogen can be used individually or as a mixture.
[0064] Phosphoric acid, potassium dihydrogen phosphate, dipotassium
hydrogen phosphate or the corresponding sodium-containing salts can
be used as the source of phosphorus. The culture medium must
furthermore comprise salts of metals, such as magnesium sulfate or
iron sulfate, which are necessary for growth.
[0065] Finally, essential growth substances, such as amino acids
and vitamins, can be employed in addition to the above-mentioned
substances. Suitable precursors can be added to the culture medium
in the form of a single batch, or can be fed in during culture.
[0066] Basic compounds, such as sodium hydroxide, potassium
hydroxide, ammonia or aqueous ammonia, or acid compounds, such as
phosphoric acid or sulfuric acid, can be used to control the pH of
the culture. Antifoams, such as, e.g., fatty acid polyglycol
esters, can be employed to control the development of foam.
Substances such as, e.g., antibiotics, can be added to the medium
to maintain the stability of plasmids and oxygen or
oxygen-containing gas mixtures, such as, e.g., air, can be
introduced into the culture to maintain aerobic conditions. The
temperature of the culture should typically be 20.degree. C. to
45.degree. C., and preferably 25.degree. C. to 40.degree. C.
Culturing is continued until a maximum of the desired product has
formed. This target is usually reached within 10 hours to 160
hours.
[0067] Methods for assaying L-amino acids are well known in the
art. Analysis can, for example, be carried out as described by
Spackman et al. (Analyt. Chem. 30:1190 (1958)) by anion exchange
chromatography with subsequent ninhydrin derivation, or by reversed
phase HPLC, for example as described by Lindroth et al. (Analyt.
Chem. 51:1167-1174 (1979)).
[0068] The invention may be further understood by reference to the
following non-limiting examples.
EXAMPLES
Example 1
Deletion of the mez Gene
[0069] Chromosomal DNA is isolated from the strain ATCC13032 by the
method of Tauch, et al. (Plasmid 33:168-179 (1995)). On the basis
of the sequence of the mez gene known for C. glutamicum from the
patent application EP1108790 (sequence no. 3328 and sequence no.
7069); from patent application WO 0100844 (sequence no. 577 under
Identification Code RXN10148); and from the databank of the
National Center for Biotechnology Information (NCBI) of the
National Library of Medicine (Bethesda, Md., USA, under Accession
Number AX123412, Accession Number AX127153 and Accession Number
AX065451), the following oligonucleotides are chosen for generation
of the mez deletion allele by means of the polymerase chain
reaction (PCR) using the "Splicing by Overlap Extension" method
(Gene SOEing Method) (Horton, Mol. Biotech. 3:93-98 (1995)):
2 Primer mez_1: 5'-ATG ACC ATC GAC CTG CAG CG-3' (SEQ ID NO:3)
Primer mez_2: 5'-AAGAAGGCGCGATGGCTGCG-3' (SEQ ID NO:4) Primer
mez_3: 5'-CGC AGC CAT CGC GCC TTC TTA ATG AGG CTT TCA CCG GCG C-3'
(SEQ ID NO:5) Primer mez_4: 5'-AAG CGT TTT GCG CTT CGG CG-3' (SEQ
ID NO:6)
[0070] The primers shown are synthesized by MWG Biotech (Ebersberg,
Germany) and the PCR reaction is carried out using Pfu polymerase
(Stratagene, product no. 600135, La Jolla, USA) and a PTC 100
Thermocycler (MJ Research Inc., Waltham, USA). The primer
mez.sub.--3 is composed of two regions of the nucleotide sequence
which bind to nucleotides 384 to 403 and 766 to 785 within the
coding sequence of mez.
[0071] Using the polymerase chain reaction, the primers mez.sub.--1
and mez.sub.--2 allow amplification of a DNA fragment 403 bp in
size and the primers mez.sub.--3 and mez.sub.--4 allow
amplification of a DNA fragment 432 bp in size. The amplification
products are tested electrophoretically in a 0.8% agarose gel,
isolated from the agarose gel with the High Pure PCR Product
Purification Kit (product no. 1732676, Roche Diagnostics GmbH,
Mannheim, Germany) and employed together as templates for a further
PCR reaction with the primers mez.sub.--1 and mez.sub.--4. The mez
deletion derivative 815 bp in size is generated in this manner (SEQ
ID NO:7). The product amplified in this way is tested
electrophoretically in a 0.8% agarose gel.
Example 2
Cloning of the mez Deletion Derivative in the Vector pCRBluntII
[0072] The amplified DNA fragment of 815 bp length which carries
the mez deletion derivative is ligated with the Zero Blunt.TM. Kit
of Invitrogen Corporation (Carlsbad, Calif., USA; Catalogue Number
K2700-20) in the vector pCR.RTM.Blunt II (Bernard, et al., J. Mol.
Biol. 234:534-541 (1993)). The E. coli strain Top10 (Grant, et al.,
Proc. Nat'l Acad. Sci. USA 87:4645-4649 (1990)) is then transformed
with the ligation batch in accordance with the instructions of the
manufacturer of the kit (Invitrogen Corporation, Carlsbad, Calif.,
USA). Selection for plasmid-carrying cells is made by plating out
the transformation batch on LB agar (Sambrook et al., Molecular
Cloning: A Laboratory Manual., 2.sup.nd ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 1989), supplemented
with 25 mg/l kanamycin. Plasmid DNA is isolated from a transformant
with the aid of the QIAprep Spin Miniprep Kit from Qiagen (Hilden,
Germany) and checked by treatment with the restriction enzymes
EcoRV and EcoRI followed by agarose gel electrophoresis (0.8%). The
plasmid is called pCRBlunt_delmez.
Example 3
Construction of the Exchange Vector pK18mobsacBdeltamez
[0073] The mez deletion derivative is isolated from the plasmid
pCRBlunt_delmez described in Example 2 by complete cleavage with
the enzyme EcoRI. After separation in an agarose gel (0.8%) with
the High Pure PCR Product Purification Kit (product no. 1732676,
Roche Diagnostics GmbH, Mannheim, Germany), fragment approximately
0.83 kb in size carrying the mez deletion derivative is isolated
from the agarose gel.
[0074] The mez deletion derivative obtained in this way is employed
for ligation with the mobilizable cloning vector pK18mobsacB
(Schfer, et al., Gene 14:69-73 (1994)). This is cleaved completely
beforehand with the restriction endonuclease EcoRI and subsequently
dephosphorylated with shrimp alkaline phosphatase (Roche
Diagnostics GmbH, Mannheim, Germany, Product Description SAP,
product no. 1758250). The vector prepared in this way is mixed with
the mez deletion allele and the mixture is treated with T4 DNA
ligase (Amersham-Pharmacia, Freiburg, Germany).
[0075] The E. coli strain DH5.alpha.mcr (Grant, Proc. Nat'l Acad.
Sci. USA 87:4645-4649 (1990)) is then electroporated with the
ligation batch (Hanahan, In. DNA Cloning, A Practical Approach,
vol. 1, ILR-Press, Cold Spring Harbor, N.Y., 1989). Selection of
plasmid-carrying cells is made by plating out the transformation
batch on LB agar (Sambrook et al., Molecular Cloning: A Laboratory
Manual, 2.sup.nd ed., Cold Spring Harbor, N.Y., 1989), which has
been supplemented with 25 mg/l kanamycin.
[0076] Plasmid DNA is isolated from a transformant with the aid of
the QIAprep Spin Miniprep Kit from Qiagen and the cloned mez
deletion allele is verified by means of restriction cleavage with
the restriction endonucleases EcoRI and BamHI. The plasmid is
called pK18mobsacBdeltamez and is shown in FIG. 1. The strain is
called E. coli DH5.alpha.mcr/pK18mobsacBdeltamez.
Example 4
Deletion Mutagenesis of the mez Gene in the C. glutamicum Strain
DM1637
[0077] The Corynebacterium glutamicum strain DM1637 is prepared by
multiple, non-directed mutagenesis, selection and mutant selection
from Corynebacterium glutamicum ATCC21527. The strain is resistant
to the lysine analogue S-(2-aminoethyl)-L-cysteine and auxotrophic
for the amino acids L-methionine and L-threonine.
[0078] The vector pK18mobsacBdeltamez described in Example 3 is
transferred by means of conjugation using the protocol of Schfer et
al. (J. Microbiol. 172:1663-1666) (1990)) into the Corynebacterium
glutamicum strain DM1637. The vector cannot replicate independently
in this strain and is retained in the cells only if it has
integrated into the chromosome as the consequence of a
recombination event. Selection of clones with integrated
pK18mobsacBdeltamez is carried out by plating the conjugation batch
on LB agar (Sambrook et al., Molecular Cloning: A Laboratory
Manual, 2.sup.nd ed., Cold Spring Habor, N.Y., 1989), which has
been supplemented with 15 mg/l kanamycin and 50 mg/ml nalidixic
acid. Clones which grow are plated on LB agar plates with 25 mg/l
kanamycin and incubated for 16 hours at 33.degree. C. For selection
of mutants in which excision of the plasmid has taken place as a
consequence of a second recombination event, the clones are
cultured unselectively for 20 hours in LB liquid medium and then
plated out on LB agar with 10% sucrose and incubated for 24
hours.
[0079] The plasmid pK18mobsacBdeltamez, like the starting plasmid
pK18mobsacB, contains, in addition to the kanamycin resistance
gene, a copy of the sacB gene which codes for levan sucrase from
Bacillus subtilis. Expression can be induced by sucrose. This leads
to the formation of levan sucrase which catalyses the synthesis of
the product levan, which, in turn, is toxic to C. glutamicum. Only
those clones in which the integrated pK18mobsacBdeltamez has been
excised therefore grow on LB agar with sucrose. In the excision,
either the complete chromosomal copy of the mez gene or an
incomplete copy with the internal deletion is removed.
[0080] Approximately 40 to 50 colonies are tested for the phenotype
"growth in the presence of sucrose" and "non-growth in the presence
of kanamycin." To demonstrate that the deleted mez allele has
remained in the chromosome, approximately 20 colonies which show
the phenotype "growth in the presence of sucrose" and "non-growth
in the presence of kanamycin" are investigated with the aid of the
polymerase chain reaction by the standard PCR method of Innis, et
al. (PCR protocols, A Guide to Methods and Applications, Academic
Press, 1990). A DNA fragment which carries the mez gene and
surrounding regions is amplified from the chromosomal DNA of the
colonies using the following primers:
3 mez-A1: 5'agt agc agc cca aat tca gc 3' (SEQ ID NO:8) mez-E1:
5'ggg cct caa gtt tgc tct ta 3' (SEQ ID NO:9)
[0081] The primers allow amplification of a DNA fragment
approximately 1.5 kb in size in control clones with the complete
mez allele. In clones with a deleted mez allele, DNA fragments with
a size of approximately 1.14 kb are amplified. The amplified DNA
fragments are identified by means of electrophoresis in a 0.8%
agarose gel. It could thus be demonstrated that the strain DM1637
carries a deleted mez allele in its chromosome. The strain was
called C. glutamicum DM1637deltamez.
Example 5
Preparation of Lysine
[0082] The C. glutamicum strain DM1637deltamez obtained in Example
4 is cultured in a nutrient medium suitable for the production of
lysine and the lysine content in the culture supernatant is
determined. To accomplish this, the strain is first incubated on an
agar plate for 24 hours at 33.degree. C. Starting from this agar
plate culture, a preculture is seeded (10 ml MM medium in a 100 ml
conical flask) and incubated for 24 hours at 33.degree. C. at 240
rpm on a shaking machine. A main culture is seeded from this
preculture such that the initial OD (660 nm) of the main culture is
0.1. MM medium, as shown below, is also used for the main
culture.
4 Medium MM CSL 5 g/l MOPS 20 g/l Glucose (autoclaved separately)
50 g/l Salts: (NH.sub.4).sub.2SO.sub.4 25 g/l KH.sub.2PO.sub.4 0.1
g/l MgSO.sub.4 * 7 H.sub.2O 1,0 g/l CaCl.sub.2 * 2 H.sub.2O 10 mg/l
FeSO.sub.4 * 7 H.sub.2O 10 mg/l MnSO.sub.4 * H.sub.2O 5.0 mg/l
Biotin (sterile-filtered) 0.3 mg/l Thiamine * HCl
(sterile-filtered) 0.2 mg/l L-Homoserine (sterile-filtered) 0.4 g/l
CaCO.sub.3 25 g/l
[0083] The CSL (corn steep liquor), MOPS (morpholinopropanesulfonic
acid) and the salt solution are brought to pH 7 with aqueous
ammonia and autoclaved. The sterile substrate and vitamin
solutions, as well as the CaCO.sub.3 autoclaved in the dry state,
are then added. Culturing is carried out in a 10 ml volume in a 100
ml conical flask with baffles at 33.degree. C. and 80% atmospheric
humidity. After 48 hours, the OD is determined at a measurement
wavelength of 660 nm with a Biomek 1000 (Beckmann Instruments GmbH,
Munich). The amount of lysine formed is determined with an amino
acid analyzer from Eppendorf-BioTronik (Hamburg, Germany) by ion
exchange chromatography and post-column derivation with ninhydrin
detection. The result of the experiment is shown in Table 1.
5 TABLE 1 GD Lysine HCl Strain (660 nm) g/l DM1637 10.9 11.3
DM1637deltamez 10.8 11.8
[0084] All references cited herein are fully incorporated by
reference. Having now fully described the invention, it will be
understood by those of skill in the art that the invention may be
performed within a wide and equivalent range of conditions,
parameters and the like, without affecting the spirit or scope of
the invention or any embodiment thereof.
Sequence CWU 1
1
9 1 1179 DNA Corynebacterium glutamicum CDS (1)..(1176) mez gene 1
atg acc atc gac ctg cag cgt tcc acc caa aac ctc acc cat gag gaa 48
Met Thr Ile Asp Leu Gln Arg Ser Thr Gln Asn Leu Thr His Glu Glu 1 5
10 15 atc ttc gag gca cac gag ggc gga aag ctc tcc att agt tcc act
cgt 96 Ile Phe Glu Ala His Glu Gly Gly Lys Leu Ser Ile Ser Ser Thr
Arg 20 25 30 ccg ctc cgc gac atg cgc gat ctt tcc ctt gct tac acc
cct ggt gtt 144 Pro Leu Arg Asp Met Arg Asp Leu Ser Leu Ala Tyr Thr
Pro Gly Val 35 40 45 gct cag gtt tgt gaa gca atc aag gaa gat cca
gag gtt gcg cgc acc 192 Ala Gln Val Cys Glu Ala Ile Lys Glu Asp Pro
Glu Val Ala Arg Thr 50 55 60 cac acg ggc att gga aac acc gtc gcg
gtt att tcc gac ggc acc gct 240 His Thr Gly Ile Gly Asn Thr Val Ala
Val Ile Ser Asp Gly Thr Ala 65 70 75 80 gtt ctt ggc ctt ggc gat atc
gga cct cag gcc tcc ctt ccc gtc atg 288 Val Leu Gly Leu Gly Asp Ile
Gly Pro Gln Ala Ser Leu Pro Val Met 85 90 95 gag ggc aag gct cag
ctg ttt agc tct ttc gct ggc ctg aag gct atc 336 Glu Gly Lys Ala Gln
Leu Phe Ser Ser Phe Ala Gly Leu Lys Ala Ile 100 105 110 cct atc gtt
ttg gac gtt cac gat gtt gac gct ttg gtt gag acc atc 384 Pro Ile Val
Leu Asp Val His Asp Val Asp Ala Leu Val Glu Thr Ile 115 120 125 gca
gcc atc gcg cct tct ttc ggt gct atc aac ttg gag gac atc tcc 432 Ala
Ala Ile Ala Pro Ser Phe Gly Ala Ile Asn Leu Glu Asp Ile Ser 130 135
140 gct cct cgt tgc ttc gag gtg gag cgc cgc ctc atc gag cgt ctc gat
480 Ala Pro Arg Cys Phe Glu Val Glu Arg Arg Leu Ile Glu Arg Leu Asp
145 150 155 160 att cca gtt atg cac gat gac cag cac ggc acc gct gtg
gtt atc ctc 528 Ile Pro Val Met His Asp Asp Gln His Gly Thr Ala Val
Val Ile Leu 165 170 175 gct gcg ctg cgc aac tcc ctg aag ctg ctg gat
cgc aag atc gaa gac 576 Ala Ala Leu Arg Asn Ser Leu Lys Leu Leu Asp
Arg Lys Ile Glu Asp 180 185 190 ctc aag att gtt att tcc ggc gca ggc
gca gcg ggc gtt gca gct gta 624 Leu Lys Ile Val Ile Ser Gly Ala Gly
Ala Ala Gly Val Ala Ala Val 195 200 205 gat atg ctg acc aac gct gga
gca acc gac atc gtg gtt ctt gat tcc 672 Asp Met Leu Thr Asn Ala Gly
Ala Thr Asp Ile Val Val Leu Asp Ser 210 215 220 cga ggc atc atc cac
gac agc cgt gag gat ctt tcc cca gtt aag gct 720 Arg Gly Ile Ile His
Asp Ser Arg Glu Asp Leu Ser Pro Val Lys Ala 225 230 235 240 gct ctt
gca gag aag acc aac cct cgt ggc atc agc ggt ggc atc aat 768 Ala Leu
Ala Glu Lys Thr Asn Pro Arg Gly Ile Ser Gly Gly Ile Asn 245 250 255
gag gct ttc acc ggc gcg gac ctg ttc att ggc gtg tcc ggc ggc aac 816
Glu Ala Phe Thr Gly Ala Asp Leu Phe Ile Gly Val Ser Gly Gly Asn 260
265 270 atc ggc gag gac gct ctc aaa ctc atg gcc ccg gag cca atc ctg
ttc 864 Ile Gly Glu Asp Ala Leu Lys Leu Met Ala Pro Glu Pro Ile Leu
Phe 275 280 285 acc ctg gcg aac cca acc cca gag atc gat cct gag ctg
tct cag aag 912 Thr Leu Ala Asn Pro Thr Pro Glu Ile Asp Pro Glu Leu
Ser Gln Lys 290 295 300 tac ggc gcc atc gtc gcg acc ggc cgc tct gac
ctg cct aac cag atc 960 Tyr Gly Ala Ile Val Ala Thr Gly Arg Ser Asp
Leu Pro Asn Gln Ile 305 310 315 320 aac aac gtg ctc gcg ttc cca gga
att ttc gcc ggc gct ctc gca gcc 1008 Asn Asn Val Leu Ala Phe Pro
Gly Ile Phe Ala Gly Ala Leu Ala Ala 325 330 335 aag gct aag aag atc
acc ccc gag atg aag ctc gcc gct gca gag gca 1056 Lys Ala Lys Lys
Ile Thr Pro Glu Met Lys Leu Ala Ala Ala Glu Ala 340 345 350 atc gcc
gac atc gca gct gag gac ctc gag gtc ggc cgc atc gtg cct 1104 Ile
Ala Asp Ile Ala Ala Glu Asp Leu Glu Val Gly Arg Ile Val Pro 355 360
365 acc gcc ctg gat ccc cgc gtc gcc cca gca gtc aag gca gct gtc cag
1152 Thr Ala Leu Asp Pro Arg Val Ala Pro Ala Val Lys Ala Ala Val
Gln 370 375 380 gcc gtc gcc gaa gcg caa aac gct taa 1179 Ala Val
Ala Glu Ala Gln Asn Ala 385 390 2 392 PRT Corynebacterium
glutamicum 2 Met Thr Ile Asp Leu Gln Arg Ser Thr Gln Asn Leu Thr
His Glu Glu 1 5 10 15 Ile Phe Glu Ala His Glu Gly Gly Lys Leu Ser
Ile Ser Ser Thr Arg 20 25 30 Pro Leu Arg Asp Met Arg Asp Leu Ser
Leu Ala Tyr Thr Pro Gly Val 35 40 45 Ala Gln Val Cys Glu Ala Ile
Lys Glu Asp Pro Glu Val Ala Arg Thr 50 55 60 His Thr Gly Ile Gly
Asn Thr Val Ala Val Ile Ser Asp Gly Thr Ala 65 70 75 80 Val Leu Gly
Leu Gly Asp Ile Gly Pro Gln Ala Ser Leu Pro Val Met 85 90 95 Glu
Gly Lys Ala Gln Leu Phe Ser Ser Phe Ala Gly Leu Lys Ala Ile 100 105
110 Pro Ile Val Leu Asp Val His Asp Val Asp Ala Leu Val Glu Thr Ile
115 120 125 Ala Ala Ile Ala Pro Ser Phe Gly Ala Ile Asn Leu Glu Asp
Ile Ser 130 135 140 Ala Pro Arg Cys Phe Glu Val Glu Arg Arg Leu Ile
Glu Arg Leu Asp 145 150 155 160 Ile Pro Val Met His Asp Asp Gln His
Gly Thr Ala Val Val Ile Leu 165 170 175 Ala Ala Leu Arg Asn Ser Leu
Lys Leu Leu Asp Arg Lys Ile Glu Asp 180 185 190 Leu Lys Ile Val Ile
Ser Gly Ala Gly Ala Ala Gly Val Ala Ala Val 195 200 205 Asp Met Leu
Thr Asn Ala Gly Ala Thr Asp Ile Val Val Leu Asp Ser 210 215 220 Arg
Gly Ile Ile His Asp Ser Arg Glu Asp Leu Ser Pro Val Lys Ala 225 230
235 240 Ala Leu Ala Glu Lys Thr Asn Pro Arg Gly Ile Ser Gly Gly Ile
Asn 245 250 255 Glu Ala Phe Thr Gly Ala Asp Leu Phe Ile Gly Val Ser
Gly Gly Asn 260 265 270 Ile Gly Glu Asp Ala Leu Lys Leu Met Ala Pro
Glu Pro Ile Leu Phe 275 280 285 Thr Leu Ala Asn Pro Thr Pro Glu Ile
Asp Pro Glu Leu Ser Gln Lys 290 295 300 Tyr Gly Ala Ile Val Ala Thr
Gly Arg Ser Asp Leu Pro Asn Gln Ile 305 310 315 320 Asn Asn Val Leu
Ala Phe Pro Gly Ile Phe Ala Gly Ala Leu Ala Ala 325 330 335 Lys Ala
Lys Lys Ile Thr Pro Glu Met Lys Leu Ala Ala Ala Glu Ala 340 345 350
Ile Ala Asp Ile Ala Ala Glu Asp Leu Glu Val Gly Arg Ile Val Pro 355
360 365 Thr Ala Leu Asp Pro Arg Val Ala Pro Ala Val Lys Ala Ala Val
Gln 370 375 380 Ala Val Ala Glu Ala Gln Asn Ala 385 390 3 20 DNA
Corynebacterium glutamicum misc_feature (1)..(20) Primer mez_1 3
atgaccatcg acctgcagcg 20 4 20 DNA Corynebacterium glutamicum
misc_feature (1)..(20) Primer mez_2 4 aagaaggcgc gatggctgcg 20 5 40
DNA Artificial sequence misc_feature (1)..(40) Primer mez_3 5
cgcagccatc gcgccttctt aatgaggctt tcaccggcgc 40 6 20 DNA
Corynebacterium glutamicum misc_feature (1)..(20) Primer mez_4 6
aagcgttttg cgcttcggcg 20 7 815 DNA Artificial sequence misc_feature
(1)..(815) PCR product mez deletion derivative 7 atgaccatcg
acctgcagcg ttccacccaa aacctcaccc atgaggaaat cttcgaggca 60
cacgagggcg gaaagctctc cattagttcc actcgtccgc tccgcgacat gcgcgatctt
120 tcccttgctt acacccctgg tgttgctcag gtttgtgaag caatcaagga
agatccagag 180 gttgcgcgca cccacacggg cattggaaac accgtcgcgg
ttatttccga cggcaccgct 240 gttcttggcc ttggcgatat cggacctcag
gcctcccttc ccgtcatgga gggcaaggct 300 cagctgttta gctctttcgc
tggcctgaag gctatcccta tcgttttgga cgttcacgat 360 gttgacgctt
tggttgagac catcgcagcc atcgcgcctt cttaatgagg ctttcaccgg 420
cgcggacctg ttcattggcg tgtccggcgg caacatcggc gaggacgctc tcaaactcat
480 ggccccggag ccaatcctgt tcaccctggc gaacccaacc ccagagatcg
atcctgagct 540 gtctcagaag tacggcgcca tcgtcgcgac cggccgctct
gacctgccta accagatcaa 600 caacgtgctc gcgttcccag gaattttcgc
cggcgctctc gcagccaagg ctaagaagat 660 cacccccgag atgaagctcg
ccgctgcaga ggcaatcgcc gacatcgcag ctgaggacct 720 cgaggtcggc
cgcatcgtgc ctaccgccct ggatccccgc gtcgccccag cagtcaaggc 780
agctgtccag gccgtcgccg aagcgcaaaa cgctt 815 8 20 DNA Corynebacterium
glutamicum misc_feature (1)..(20) Primer mez-A1 8 agtagcagcc
caaattcagc 20 9 20 DNA Corynebacterium glutamicum misc_feature
(1)..(20) Primer mez-E1 9 gggcctcaag tttgctctta 20
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